The long-term objective of the proposed protect the retinal pigment epithelium (RPE) from oxidative stress. Oxidative injury to the aged RPE may reduce its ability to support retinal photoreceptors, contributing to diseases like age-related macular degeneration. This application focuses on the pigment melanin. Melanin, which is a hallmark feature of RPE cells, can show antioxidant properties under some conditions, although melanin has not been carefully investigated to determine whether it actually protects cells from oxidative stress. The melanin of RPE cells is unusual in that it shows little turnover after embryogenesis and it may, therefore, be susceptible to changes with age. The underlying hypothesis of this application is that melanin protects RPE cells from oxidative stress induced in the outer retina by blue light, redox active metal ions and reactive oxygen species, but aging modifies melanin's physicochemical properties, reducing its efficiency as an antioxidant. In this project a cell biologist and a biophysicist plan to collaborate, using sensitive electron spin resonance (ESR) methods, cell culture models, and several biochemical and morphologic measures of oxidative stress and cytotoxicity, to address these specific aims: (1) To determine whether bovine or human RPE melanin inhibits lipid peroxidation, whether aging affects this property and whether the mechanism of melanin's putative antioxidant function can be identified by experimentally modifying the pigment granules. (2) To introduce intact or experimentally modified melanin granules by phagocytosis into a cultured human RPE cell line (ARPE-19 cells) to determine whether melanin functions as an antioxidant within cells and protects cells from photically- or chemically-induced oxidative stress. (3) To determine whether the antioxidant and cyto-protective properties of melanin from young and aged human donors differs in oxidatively-stressed cells, and whether cellular aging of the RPE, using the model of replicative senescence, modulates melanin's antioxidant function. Detailed analysis of the physicochemical properties of melanin, coupled with testing of its biological functions within RPE cells, will provide fundamental information about an important class of RPE granules whose properties may change with age.